Viral pathogens possessing nonsegmented, negative-sense RNA genomes are the cause of devastating diseases worldwide (e.g. Ebola, measles, and rabies viruses). Virion assembly and release (budding) are important late events in the replication cycles of these viruses; however, the molecular mechanisms employed by negative-sense RNA viruses to bud and separate from cells remain largely undefined. The experiments described in this proposal are significant for the following reasons: i) They will provide fundamental information on the mechanisms of virus budding (Sp. Aim 1). ii) They not only will provide information about the virus, but also may provide information concerning the role of the host cell in virus budding (Sp. Aim 2). iii) They may be applicable for the development of future DNA-based vaccines (Sp. Aim 3). A highly conserved proline-rich motif (PY motif) within the M protein of vesicular stomatitis virus (VSV) has been implicated in mediating both release of M protein from mammalian cells and interactions with specific domains (WW-domains) of cellular proteins. The biological significance of budding domains within the VSV M protein will be tested using reverse-genetics techniques and electron microscopic analysis. Virus-host interactions mediated by the viral PY motif and the cellular WW-domain will be characterized both in vitro and in vivo. The biological relevance of these protein-protein interactions will be assessed in both yeast and mammalian cells. Lastly, the ability of the PY motif from the VSV M protein to direct the budding of an otherwise nonbudding, heterologous protein will be tested in a functional budding assay. Overall, the experiments described in this proposal not only will provide important insights into the molecular aspects of budding of rhabdoviruses, but also will likely provide important insights into the budding mechanisms of other negative-sense RNA viruses that possess conserved PY or PY-like motifs.